Difference between revisions of "PCR"
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(Created page with "For a 25 ul reaction add: :H<sub>2</sub>O 10.5 µl :Taq 2X Master Mix 12.5 µl :Forward primer 0.5 ul 10 µM solution :Reverse primer 0.5 ul 10 µM solution :DNA template 1...") |
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| Line 1: | Line 1: | ||
| − | For a 25 | + | For a 25 µl total volume reaction using a micropipette add to a PCR tube: |
:H<sub>2</sub>O 10.5 µl | :H<sub>2</sub>O 10.5 µl | ||
:Taq 2X Master Mix 12.5 µl | :Taq 2X Master Mix 12.5 µl | ||
| − | :Forward primer 0.5 | + | :Forward primer 0.5 µl 10 µM solution |
| − | :Reverse primer 0.5 | + | :Reverse primer 0.5 µl 10 µM solution |
| − | :DNA template 1 µl | + | :DNA template 1 µl (adding sample DNA last minimizes potential contamination of the other solutions) |
| − | General PCR cycling conditions are: | + | This is then placed in a thermocycler. General PCR cycling conditions are: |
:An initial 95 C 30 s | :An initial 95 C 30 s | ||
| Line 15: | Line 15: | ||
::70 C 1 m extension (this can be raised to 5 m for several kbp long segments) | ::70 C 1 m extension (this can be raised to 5 m for several kbp long segments) | ||
:With a final extension of 70 C for 5 minutes. | :With a final extension of 70 C for 5 minutes. | ||
| + | |||
| + | There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 2<sup>30</sup>) of a single starting DNA template molecule. | ||
Revision as of 15:41, 16 July 2018
For a 25 µl total volume reaction using a micropipette add to a PCR tube:
- H2O 10.5 µl
- Taq 2X Master Mix 12.5 µl
- Forward primer 0.5 µl 10 µM solution
- Reverse primer 0.5 µl 10 µM solution
- DNA template 1 µl (adding sample DNA last minimizes potential contamination of the other solutions)
This is then placed in a thermocycler. General PCR cycling conditions are:
- An initial 95 C 30 s
- Then 30 cycles of:
- 95 C 20 s melting
- annealing temperature (this varies with the primers used, usually between 55 C and 65 C although some primers can be lower) 40s
- 70 C 1 m extension (this can be raised to 5 m for several kbp long segments)
- With a final extension of 70 C for 5 minutes.
There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 230) of a single starting DNA template molecule.
